Nucleotide encoding canine 5T4 antigen

ABSTRACT

Canine and feline 5T4 polypeptide sequences and nucleotide sequences encoding them are provided. A vector system comprising a nucleic acid encoding 5T4 and a 5T4-specific agent are also provided.

RELATED APPLICATIONS

This application is a national phase application under 35 U.S.C. §371 ofPCT/GB01/05004, filed on Nov. 13, 2001, designating the U.S., publishedon May 16, 2002 as WO 02/38612, and claiming priority from InternationalApplication No. PCT/GB00/04317, filed on Nov. 13, 2000. All of theabove-mentioned applications, as well as all documents cited herein, anddocuments referenced or cited in documents cited herein, areincorporated by reference.

FIELD OF THE INVENTION

The present invention relates to 5T4 antigens, 5T4-specific agents andtheir use in immunotherapy.

BACKGROUND TO THE INVENTION

Tumors are relatively common in companion animals (see Mailto andLagadic (1990) Recueil de Medacine. Veterinaire Special Cancerologiep937-947 for a descriptive epidemiology of canine and feline tumors).

Current methods for treating tumors in cats and dogs include surgery,chemotherapy and radiotherapy. These methods are associated with anumber of disadvantages. For example, they all involve a high level oftrauma for the patient and they are not always effective in eliminatingall the cancerous cells.

An alternative therapeutic approach is immunotherapy which involves thespecific or non-specific stimulation of immune reactions of the patientin order to promote the immunological rejection of cancerous cells.There are several methods of immunotherapy:

Non-specific immunotherapy—non-specific stimulation of the immune system(e.g. by using an agent which acts like an adjuvant).

Specific Passive immunotherapy (serotherapy)—transferring anti-tumorantibodies to the patient.

Adoptive immunotherapy—transferring immunocompetent allogenic cells froma healthy individual (eg bone marrow cells)

Specific active immunotherapy—stimulating the immune defenses of thecancer patient by providing the antigens associated with the tumor (e.g.using irradiated cancer cells)

Immunotherapy may be a method of complementary treatment used incombination with surgery, chemotherapy and/or radiotherapy. A study ofimmunotherapeutic chemical trials in cats and dogs is given in Hayes(1990) Recueil de Medicine Veterinaire 16(11).

An immunotherapeutic approach may be directed against an antigen whichis peculiar to the tumor. One strategy for canine and feline cancerimmunotherapy would be to identify a tumor-associated antigens (TAAs),expressed on cat or dog tumors, useful for eliciting an anti-tumorimmunotherapeutic response.

SUMMARY OF THE INVENTION

The present inventors have shown that a significant proportion of canineand feline tumors express an oncofetal leucine-rich glycoprotein, knownas “5T4”. There is also presented for the first time the full proteinand nucleotide sequences for canine 5T4 and feline 5T4.

The first aspect of the invention relates to canine and feline 5T4sequences.

In this aspect, the present invention provides

-   -   i) a canine 5T4 polypeptide having the amino acid sequence shown        in SEQ ID No 1 or a variant, homologue, fragment or derivative        thereof; and    -   ii) a nucleotide sequence capable of encoding such a canine 5T4        polypeptide. Preferably the nucleotide sequence has the sequence        shown as SEQ ID NO 2 or a variant, homologue, fragment or        derivative thereof.

The present invention also provides

-   -   i) a feline 5T4 polypeptide having the amino acid sequence shown        in SEQ ID No 3 or a variant, homologue, fragment or derivative        thereof; and    -   ii) a nucleotide sequence capable of encoding such a feline 5T4        polypeptide. Preferably the nucleotide sequence has the sequence        shown as SEQ ID NO 4 or a variant, homologue, fragment or        derivative thereof.

The present invention also provides an agent specific to a canine orfeline 0.5T4 nucleotide sequence. For example, the agent may comprise anantisense sequence capable of binding specifically to the 5T4 sequence.

In a second aspect, the present invention provides a vector systemexpressing a polynucleotide encoding a canine or feline 5T4 antigen.

Expression of 5T4 antigen in a subject elicits an immunotherapeuticanti-tumor response. Preferably, the viral vector favours CTL responsesto expressed antigens, and is advantageously a poxvirus vector, such asa vaccinia virus vector. Further vectors, both viral and non-viral,which are suitable for delivering 5T4 antigen are described below.

In a third aspect, the invention provides an agent capable of bindingspecifically to canine or feline 5T4 protein. The agent may be anantibody. For example, the agent may be an antibody raised against thecanine or feline 5T4 protein (or fragment thereof) of the first aspectof the invention.

The present invention also provides to the sequential use of a vectorencoding a 5T4 antigen and such an anti-5T4 agent. Where the anti-5T4agent is a protein (such as an antibody or derivative thereof) it may beadministered as naked DNA (for example, in a plasmid), or in anexpression vector (which may be viral or non-viral) or directly in aprotein form. The agent may be fused with an immunostimulatory molecule.

Thus, the invention provides a kit comprising a vector according to thesecond aspect of the invention and an agent according to the thirdaspect of the invention, for simultaneous, separate, or sequential use,preferably for use in the treatment of tumors.

In a fourth aspect the present invention provides a vaccine, primingcomposition or boosting composition comprising such a polypeptide,polynucleotide, vector system or agent. The vaccine, priming or boostingcomposition may comprise one or more adjuvants.

It has been found that multiple-dose procedures are often more effectiveat generating an immune response that a single administration ofvaccine. Prime-boost regimes may be homologous (where the samecomposition is administered two or more times) or heterologous.

An example of a heterologous prime-boost regime would be theadministration of at least one dose of a DNA vaccine, followed by atleast one dose of a viral vaccine. An example of a homologousprime-boost regime is repeated doses of a viral vector system.

In this respect, the present invention also comprises a kit whichcomprises:

-   -   a first composition comprising a polynucleotide encoding a 5T4        antigen, and    -   a second composition comprising a vector system according to the        second aspect of the invention    -   for simultaneous, separate or sequential administration to a        subject.

The first composition may, for example, be a naked DNA vector.

In a fifth aspect, the present invention provides the use of such a 5T4antigen, polypeptide, polynucleotide, vaccine, priming composition,boosting composition or kit in the manufacture of a medicament for theprevention and/or treatment of a disease in a subject.

The present invention also provides a method for the treatment and/orprevention of a disease in a subject which comprises the step ofadministration of such a 5T4 antigen, protein, polynucleotide, vaccine,priming composition, boosting composition to the subject.

Preferably the method of the fifth aspect of the invention is a methodfor the immunotherapy of a tumor in a subject.

DETAILED DESCRIPTION OF THE INVENTION

Other aspects of the present invention are presented in the accompanyingclaims and in the following description and drawings. These aspects arepresented under separate section headings. However, it is to beunderstood that the teachings under each section are not necessarilylimited to that particular section heading.

5T4 Proteins

The first aspect of the invention relates to canine and feline 5T4proteins and their associated polynucleotides.

In humans, the oncofetal leucine-rich glycoprotein, 5T4, is expressed bya wide variety of carcinomas, but on normal adult tissues expression isrestricted to the placenta with low levels also being found on a fewspecialised epithelia. Presence of the antigen on cancer cells isassociated with metastasis and has been shown to be an independentindicator of poor prognosis in a number of different cancers.

The human tumor-associated antigen 5T4 is a 72 kDa glycoprotein and hasbeen characterised (for example, in WO89/07947). The full nucleic acidsequence of human 5T4 is known (iGenBank accession no. Z29083; Myers etal., 1994 J Biol Chem 169: 9319-24).

WO 00/29428 describes the partial sequence of canine 5T4.

The present invention provides, for the first time, the full amino acidand nucleic acid sequences for canine and human 5T4. Despite the factthe human and partial canine sequences were available, isolation andcloning of the full canine and feline sequences was not straightforward.In this respect a number of attempts were made to isolate the caninegene by PCR using primers based on the human sequence immediatelyoutside the coding region. Such attempts failed due to unexpecteddifferences between the human and canine sequences immediately upstreamof the start codon.

(SEQ ID NO:5) CCCAGCTCCGGGGAGCGCCGCGCCGCGCCGCGATG Canine (SEQ ID NO:6)   AGCTCCGGGGAAACGCGAGCC failed PRIMER (SEQ ID NO:7)CCCAGCTCCGGGGAAACGCGAGCCGCGATG HumanAmino Acid Sequences

As used herein, the term “amino acid sequence” refers to peptide,polypeptide sequences, protein sequences or portions thereof.

The present invention covers variants, homologues or derivatives of theamino acid sequences presented herein, as well as variants, homologuesor derivatives of the nucleotide sequence coding for those amino acidsequences.

In the context of the present invention, a homologous sequence is takento include an amino acid sequence which is at least 75, 85 or 90%identical, preferably at least 95 or 98% identical at the amino acidlevel over at least, for example, the amino acid sequence as set out inSEQ ID No 1 or SEQ ID No 3 of the sequence listing herein. Inparticular, homology should typically be considered with respect tothose regions of the sequence known to be essential for bindingspecificity (such as amino acids at positions) rather than non-essentialneighbouring sequences. Although homology can also be considered interms of similarity (i.e. amino acid residues having similar chemicalproperties/functions), in the context of the present invention it ispreferred to express homology in terms of sequence identity.

Homology comparisons can be conducted by eye, or more usually, with theaid of readily available sequence comparison programs. Thesecommercially available computer programs can calculate % homologybetween two or more sequences.

% homology may be calculated over contiguous sequences, i.e. onesequence is aligned with the other sequence and each amino acid in onesequence is directly compared with the corresponding amino acid in theother sequence, one residue at a time. This is called an “ungapped”alignment. Typically, such ungapped alignments are performed only over arelatively short number of residues.

Although this is a very simple and consistent method, it fails to takeinto consideration that, for example, in an otherwise identical pair ofsequences, one insertion or deletion will cause the following amino acidresidues to be put out of alignment, thus potentially resulting in alarge reduction in % homology when a global alignment is performed.Consequently, most sequence comparison methods are designed to produceoptimal alignments that take into consideration possible insertions anddeletions without penalising unduly the overall homology score. This isachieved by inserting “gaps” in the sequence alignment to try tomaximise local homology.

However, these more complex methods assign “gap penalties” to each gapthat occurs in th alignment so that, for the same number of identicalamino acids, a sequence alignment with as few gaps aspossible—reflecting higher relatedness between the two comparedsequences—will achieve a higher score than one with many gaps. “Affinegap costs” are typically used that charge a relatively high cost for theexistence of a gap and a smaller penalty for each subsequent residue inthe gap. This is the most commonly used gap scoring system. High gappenalties will of course produce optimised alignments with fewer gaps.Most alignment programs allow the gap penalties to be modified. However,it is preferred to use the default values when using such software forsequence comparisons. For example when using the GCG Wisconsin Bestfitpackage (see below) the default gap penalty for amino acid sequences is−12 for a gap and −4 for each extension.

Calculation of maximum % homology therefore firstly requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (University of Wisconsin,U.S.A.; Devereux et al., 1984, Nucleic Acids Research 12:387). Examplesof other software than can perform sequence comparisons include, but arenot limited to, the BLAST package (see Ausubel et al., 1999 ibid—Chapter18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and theGENEWORKS suite of comparison tools. Both BLAST and FASTA are availablefor offline and online searching (see Ausubel et al., 1999 ibid, pages7-58 to 7-60). However it is preferred to use the GCG Bestfit program. Anew tool, called BLAST 2 Sequences is also available for comparingprotein and nucleotide sequence (se FEMS Microbiol Lett 1999 174(2):247-50; FEMS Microbiol Lett 1999 177(1): 187-8 andtatiana@ncbi.nlm.nih.gov).

Although the final % homology can be measured in terms of identity, thealignment process itself is typically not based on an all-or-nothingpair comparison. Instead, a scaled similarity score matrix is generallyused that assigns scores to each pairwise comparison based on chemicalsimilarity or evolutionary distance. An example of such a matrixcommonly used is the BLOSUM62 matrix—the default matrix for the BLASTsuite of programs. GCG Wisconsin programs generally use either thepublic default values or a custom symbol comparison table if supplied(see user manual for further details). It is preferred to use the publicdefault values for the GCG package, or in the case of other software,the default matrix, such as BLOSUM62.

Once the software has produced an optimal alignment, it is possible tocalculate % homology, preferably % sequence identity. The softwaretypically does this as part of the sequence comparison and generates anumerical result.

The terms “variant” or “derivative” in relation to the amino acidsequences of the present invention includes any substitution of,variation of, modification of, replacement of, deletion of or additionof one (or more) amino acids from or to the sequence providing theresultant amino acid sequence has a binding specificity, preferablyhaving at least the same binding specificity as the amino acid sequenceset out in SEQ ID No 1 or SEQ ID No 3 of the sequence listing herein.

SEQ ID No 1 or SEQ ID No 3 of the sequence listing herein may bemodified for use in the present invention. Typically, modifications aremade that maintain the binding specificity of the sequence. Amino acidsubstitutions may be made, for example from 1, 2 or 3 to 10 or 20substitutions provided that the modified sequence retains the requiredbinding specificity. Amino acid substitutions may include the use ofnon-naturally occurring analogues.

The 5T4 poypeptide of the present invention may also have deletions,insertions or substitutions of amino acid residues which produce asilent change and result in a functionally equivalent molecule.Deliberate amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe binding specificity of the 5T4 poypeptide is retained. For example,negatively charged amino acids include aspartic acid and glutamic acid;positively charged amino acids include lysine and arginine; and aminoacids with uncharged polar head groups having similar hydrophilicityvalues include leucine, isoleucin, valine, glycine, alanine, asparagine,glutamine, serine, threonine, phenylalanine, and tyrosine.

Conservative substitutions may be made, for example according to theTable below. Amino acids in the same block in the second column andpreferably in the same line in the third column may be substituted foreach other:

ALIPHATIC Non-polar GAP ILV Polar - uncharged CSTM NQ Polar - charged DEKR AROMATIC HFWY

Preferably, the 5T4 sequence is prepared by use of recombinanttechniques.

With regard to a fragment of the canine 5T4 sequence, preferably thefragment conprises at least one, preferably some, most preferably all ofthe amino acids 1-182 and/or 297-420 shown in SEQ ID No 1.

Nucleotide Sequences

It will be understood by a skilled person that numerous differentnucleotide sequences can encode the same 5T4 poypeptide of the presentinvention as a result of the degeneracy of the genetic code. Inaddition, it is to be understood that skilled persons may, using routinetechniques, make nucleotide substitutions that do not affect the 5T4poypeptide encoded by the nucleotide sequence of the present inventionto reflect the codon usage of any particular host organism in which the5T4 poypeptide of the present invention is to be expressed.

The terms “variant”, “homologue” or “derivative” in relation to thenucleotide sequence set out in SEQ ID No 15 (see FIG. 26) of the presentinvention includes any substitution of, variation of, modification of,replacement of, deletion of or addition of one (or more) nucleic acidfrom or to the sequence providing the resultant nucleotide sequencecodes for a canine or feline 5T4 polypeptide, preferably a polypeptideas set out in SEQ ID No 1 or 3 of the sequence listing of the presentinvention.

As indicated above, with respect to sequence homology, preferably thereis at least 75%, more preferably at least 85%, more preferably at least90% homology to the sequences shown in the sequence listing herein. Morepreferably there is at least 95%, more preferably at least 98%,homology. Nucleotide homology comparisons may be conducted as describedabove. A preferred sequence comparison program is the GCG WisconsinBestfit program described above. The default scoring matrix has a matchvalue of 10 for each identical nucleotide and −9 for each mismatch. Thedefault gap creation penalty is −50 and the default gap extensionpenalty is −3 for each nucleotide.

The present invention also encompasses nucleotide sequences that arecapable of hybridising selectively to the sequences presented herein, orany variant, fragment or derivative thereof, or to the complement of anyof the above. Nucleotide sequences are preferably at least 15nucleotides in length, more preferably at least 20, 30, 40 or 50nucleotides in length.

With regard to a fragment of the canine 5T4 sequence, preferably thefragment conprises at least one, preferably some, most preferably all ofthe nucleic acids 1-546 and/or 890-1263 shown in SEQ ID No 15.

Hybridisation

The term “hybridization” as used herein shall include “the process bywhich a strand of nucleic acid joins with a complementary strand throughbase pairing”.

Nucleotide sequences of the invention capable of selectively hybridisingto the nucleotide sequences presented herein, or to their complement,will be generally at least 75%, preferably at least 85 or 90% and morepreferably at least 95% or 98% homologous to the correspondingnucleotide sequences presented herein over a region of at least 20,preferably at least 25 or 30, for instance at least 40, 60 or 100 ormore contiguous nucleotides. Preferred nucleotide sequences of theinvention will comprise regions homologous to the nucleotide sequenceset out in SEQ ID No 2 or SEQ ID No 4 of the sequence listings of thepresent invention preferably at least 80 or 90% and more preferably atleast 95% homologous to the nucleotide sequence set out in SEQ ID. NO 2or 4 of the sequence listings of the present invention.

The term “selectively hybridizable” means that the nucleotide sequenceused as a probe is used under conditions where a target nucleotidesequence of the invention is found to hybridize to the probe at a levelsignificantly above background. The background hybridization may occurbecause of other nucleotide sequences present, for example, in the cDNAor genomic DNA library being screened. In this event, background impliesa level of signal generated by interaction between the probe and anon-specific DNA member of the library which is less than 10 fold,preferably less than 100 fold as intense as the specific interactionobserved with the target DNA. The intensity of interaction may bemeasured, for example, by radiolabelling the probe, e.g. with ³²P.

Hybridization conditions are based on the melting temperature (Tm) ofthe nucleic acid binding complex, as taught in Berger and Kimmel (1987,Guide to Molecular Cloning. Techniques, Methods in Enzymology, Vol 152,Academic Press, San Diego Calif.), and confer a defined “stringency” asexplained below.

Maximum stringency typically occurs at about Tm-5° C. (5° C. below theTm of the probe); high stringency at about 5° C. to 10° C. below Tm;intermediate stringency at about 10° C. to 20° C. below Tm; and lowstringency at about 20° C. to 25° C. below Tm. As will be understood bythose of skill in the art, a maximum stringency hybridization can beused to identify or detect identical nucleotide sequences while anintermediate (or low) stringency hybridization can be used to identifyor detect similar or related polynucleotide sequences.

In a preferred aspect, the present invention covers nucleotide sequencesthat can hybridise to the nucleotide sequence of the present inventionunder stringent conditions (e.g. 65° C. and 0.1×SSC {1×SSC=0.15 M NaCl,0.015 M Na₃ Citrate pH 7.0). Where the nucleotide sequence of theinvention is double-stranded, both strands of the duplex, eitherindividually or in combination, are encompassed by the presentinvention. Where the nucleotide sequence is single-stranded, it is to beunderstood that the complementary sequence of that nucleotide sequenceis also included within the scope of the present invention.

Nucleotide sequences which are not 100% homologous to the sequences ofthe present invention but fall within the scope of the invention can beobtained in a number of ways. Other variants of the sequences describedherein may be obtained for example by probing DNA libraries made from arange of sources. In addition, other viral/bacterial or cellularhomologues particularly cellular homologues found in mammalian cells(e.g. rat, mouse, bovine and primate cells), may be obtained and suchhomologues and fragments thereof in general will be capable ofselectively hybridising to the sequences shown in the sequence listingherein. Such sequences may be obtained by probing cDNA libraries madefrom or genomic DNA libraries from other animal species, and probingsuch libraries with probes comprising all or part of the nucleotidesequence set out in SEQ ID No 2 or SEQ ID No 4 of the sequence listingsof the present invention under conditions of medium to high stringency.Similar considerations apply to obtaining species homologues and allelicvariants of the amino acid and/or nucleotide sequences of the presentinvention.

Variants and strain/species homologues may also be obtained usingdegenerate PCR which will use primers designed to target sequenceswithin the variants and homologues encoding conserved amino acidsequences within the sequences of the present invention. Conservedsequences can be predicted, for example, by aligning the amino acidsequences from several variants/homologues. Sequence alignments can beperformed using computer software known in the art. For example the GCGWisconsin PileUp program is widely used. The primers used in degeneratePCR will contain one or more degenerate positions and will be used atstringency conditions lower than those used for cloning sequences withsingle sequence primers against known sequences.

Alternatively, such nucleotide sequences may be obtained by sitedirected mutagenesis of characterised sequences, such as the nucleotidesequence set out in SEQ ID No 2 or SEQ ID No 4 of the sequence listingsof the present invention. This may be useful where for example silentcodon changes are required to sequences to optimise codon preferencesfor a particular host cell in which the nucleotide sequences are beingexpressed. Other sequence changes may be desired in order to introducerestriction enzyme recognition sites, or to alter the activity of the5T4 poypeptide encoded by the nucleotide sequences.

The nucleotide sequences of the present invention may be used to producea primer, e.g. a PCR primer, a primer for an alternative amplificationreaction, a probe e.g. labelled with a revealing label by conventionalmeans using radioactive or non-radioactive labels, or the nucleotidesequences may be cloned into vectors. Such primers, probes and otherfragments will be at least 15, preferably at least 20, for example atleast 25, 30 or 40 nucleotides in length, and are also encompassed bythe term nucleotide sequence of the invention as used herein.

The nucleotide sequences such as a DNA polynucleotides and probesaccording to the invention may be produced recombinantly, synthetically,or by any means available to those of skill in the art. They may also becloned by standard techniques.

In general, primers will be produced by synthetic means, involving astep wise manufacture of the desired nucleic acid sequence onenucleotide at a time. Techniques for accomplishing this using automatedtechniques are readily available in the art.

Longer nucleotide sequences will generally be produced using recombinantmeans, for example using a PCR (polymerase chain reaction) cloningtechniques. This will involve making a pair of primers (e.g. of about 15to 30 nucleotides) flanking a region of the targeting sequence which itis desired to clone, bringing the primers into contact with mRNA or cDNAobtained from an animal or human cell, performing a polymerase chainreaction (PCR) under conditions which bring about amplification of thedesired region, isolating the amplified fragment (e.g. by purifying thereaction mixture on an agarose gel) and recovering the amplified DNA.The primers may be designed to contain suitable restriction enzymerecognition sites so that the amplified DNA can be cloned into asuitable cloning vector

Due to the inherent degeneracy of the genetic code, other DNA sequenceswhich encode substantially the same or a functionally equivalent aminoacid sequence, may be used to clone and express the 5T4 poypeptide. Aswill be understood by those of skill in the art, it may be advantageousto produce the 5T4 poypeptide—encoding nucleotide sequences possessingnon-naturally occurring codons. Codons preferred by a particularprokaryotic or eukaryotic host (Murray E et al(1989) Nuc Acids Res17:477-508) can be selected, for example, to increase the rate of the5T4 poypeptide expression or to produce recombinant RNA transcriptshaving desirable properties, such as a longer half-life, thantranscripts produced from naturally occurring sequence.

Agent/Antibody

The fourth aspect of the invention provides an agent capable of bindingspecifically to canine or feline 5T4 protein. The agent may be anantibody.

WO 00/29428 describes antibodies which are capable of bindingspecifically to human 5T4. The present inventors have demonstrated thatthese antibodies do not cross-react with feline or canine 5T4.

As used herein, “antibody” includes a whole immunoglobulin molecule or apart thereof or a bioisostere or a mimetic thereof or a derivativethereof or a combination thereof. Examples of a part thereof include:Fab, F(ab)′₂, and Fv. Examples of a bioisostere include single chain Fv(ScFv) fragments, chimeric antibodies, bifunctional antibodies.

The term “mimetic” relates to any chemical which may be a peptide,polypeptide, antibody or other organic chemical which has the samebinding specificity as the antibody.

The term “derivative” as used herein includes chemical modification ofan antibody. Illustrative of such modifications would be replacement ofhydrogen by an alkyl, acyl, or amino group.

A whole immunoglobulin molecule is divided into two regions: binding(Fab) domains that interact with the antigen and effector (Fc) domainsthat signal the initiation of processes such as phagocytosis. Eachantibody molecule consists of two classes of polypeptide chains, light(L) chains and heavy (H) chains. A single antibody has two indenticalcopies of the L chain and two of the H chain. The N-terminal domain fromeach chain forms the variable regions, which constitute theantigen-binding sites. The C-terminal domain is called the constantregion. The variable domains of the H (V_(H)) and L (V_(L)) chainsconstitute an Fv unit and can interact closely to form a single chain Fv(ScFv) unit. In most H chains, a hinge region is found. This hingeregion is flexible and allows the Fab binding regions to move freelyrelative to the rest of the molecule. The hinge region is also the placeon the molecule most susceptible to the action of protease which cansplit the antibody into the antigen binding site (Fab) and the effector(Fc) region.

The domain structure of the antibody molecule is favourable to proteinengineering, facilitating the exchange between molecules of functionaldomains carrying antigen-binding activities (Fabs and Fvs) or effectorfunctions (Fc). The structure of the antibody also makes it easy toproduce antibodies with an antigen recognition capacity joined tomolecules such as toxins, lymphocytes or growth factors.

Chimeric antibody technology amy involve the transplantation of antibodyvariable domains from one species (for example, a mouse) onto antibodyconstant domains from another species (for example a cat or dog).

Fab, Fv, and single chain Fv (ScFv) fragments with VH and VL joined by apolypeptide linker exhibit specificities and affinities for antigensimiliar to the original monoclonal antibodies. The ScFv fusion proteinscan be produced with a non-antibody molecule attached to either theamino or carboxy terminus. In these molecules, the Fv can be used forspecific targeting of the attached molecule to a cell expressing theappropriate antigen. Bifunctional antibodies can also be created byengineering two different binding specificities into a single antibodychain. Bifunctional Fab, Fv and ScFv antibodies may comprise engineereddomains such as CDR grafted or humanised domains.

A large number of monoclonal antibodies and immunoglobulin-likemolecules are known which bind specifically to antigens present on thesurfaces of particular cell types. Procedures for identifying,characterising, cloning and engineering these molecules are wellestablished, for example using hybridomas derived from mice ortransgenic mice, phage-display libraries or scFv libraries. Genesencoding immunoglobulins or immunoglobulin-like molecules can beexpressed in a variety of heterologous expression systems. Largeglycosylated proteins including immunoglobulins are efficiently secretedand assembled from eukaryotic cells, particularly mammalian cells.Small, non-glycosylated fragments such as Fab, Fv, or scFv fragments canbe produced in functional form in mammalian cells or bacterial cells.

The antigen-binding domain may be comprised of the heavy and lightchains of an immunoglobulin, expressed from separate genes, or may usethe light chain of an immunoglobulin and a truncated heavy chain to forma Fab or a F(ab)′₂ fragment. Alternatively, truncated forms of bothheavy and light chains may be used which assemble to form a Fv fragment.An engineered scFv fragment may also be used, in which case, only asingle gene is required to encode the antigen-binding domain.

In a preferred aspect, the present invention provides an ScFv antibody(ScFv Ab), capable of recognising canine 5T4 or feline 5T4.

The invention also provides a nucleotide sequence capable of encodingsuch an antibody or derivative thereof and a vector comprising such anucleic acid sequence. The antibody or its precursor (i.e. nucleic acidencoding the antibody) may be used in a method to treat and/or prevent adisease.

For example, an ScFv Ab can be directly administered to a cat or dogeither as a peptide (synthetically or genetically expressed) or as“naked DNA” (for example, in a plasmid) or via a delivery vehicle suchas a viral vector comprising the nucleotide sequence encoding the ScFvAb.

The antibody, or derivative thereof (for example anti-5T4 scFvs) may beused as an 5T4 targeting molecule. For example, they may be used to (i)to target natural or exogenous 5T4 in situ and/or (ii) deliver immuneenhancer molecules, such as B7.1, to natural or exogenous 5T4 in situ(Carroll et al. (1998) J Natl Cancer Inst 90(24):1881-7). Thispotentiates the immunogenicity of 5T4 in the subject.

The term “binds specifically” as used herein is intended to mean thatthe agent preferentially binds to canine and/or feline c5T4 than tohuman 5T4. Preferably the agent preferntailly binds to either canine orfeline 5T4 and does not bind or binds significantly less well to theother protein. If the agent is an antibody it may be raised againstcanine or feline 5T4 or one or more fragments thereof.

Vector Systems

The second aspect of the invention relates to a vector system expressinga polynucleotide encoding a canine or feline 5T4 antigen.

As used herein, a “vector system” may be any agent capable of deliveringor maintaining nucleic acid in a host cell, and includes viral vectors,plasmids, naked nucleic acids, nucleic acids complexed with polypeptideor other molecules and nucleic acids immobilised onto solid phaseparticles. Such vectors are described in detail below. It will beunderstood that the present invention, in its broadest form, is notlimited to any specific vector for delivery of the 5T4-encoding nucleicacid.

The 5T4 antigen is “expressed” in accordance with the present inventionby being produced in the cells of a host organism as a result oftranslation, and optionally transcription, of the nucleic acid encodingthe 5T4 antigen. Thus, 5T4 antigen is produced in situ in the cell.Since 5T4 is a transmembrane protein, the extracellular portion thereofis displayed on the surface of the cell in which it is produced. Ifnecessary, therefore, the term “expression” includes the provision ofthe necessary signals to ensure correct processing of the 5T4 antigensuch that it is displayed on the cell surface and can interact with thehost immune system.

Vector

As it is well known in the art, a vector is a tool that allows orfaciliates the transfer of an entity from one environment to another. Inaccordance with the present invention, and by way of example, somevectors used in recombinant DNA techniques allow entities, such as asegment of DNA (such as a heterologous cDNA segment, such as aheterologous cDNA segment), to be transferred into a host and/or atarget cell for the purpose of replicating the vectors comprising thenucleotide sequences of the present invention and/or expressing theproteins of the invention encoded by the nucleotide sequences of thepresent invention. Examples of vectors used in recombinant DNAtechniques include but are not limited to plasmids, chromosomes,artificial chromosomes or viruses.

“Naked DNA”

The vectors comprising nucleotide sequences encoding 5T4 poypeptide or5T4-specific agent of the present invention may be administered directlyas “a naked nucleic acid construct”, preferably further comprisingflanking sequences homologous to the host cell genome.

As used herein, the term “naked DNA” refers to a plasmid comprising anucleotide sequences encoding a 5T4 poypeptide or 5T4-specific agent ofthe present invention together with a short promoter region to controlits production. It is called “unaked” DNA because the plasmids are notcarried in any delivery vehicle. When such a DNA plasmid enters a hostcell, such as a eukaryotic cell, the proteins it encodes are transcribedand translated within the cell.

Non-Viral Delivery

Alternatively, the vectors comprising nucleotide sequences of thepresent invention may be introduced into suitable host cells using avariety of non-viral techniques known in the art, such as transfection,transformation, electroporation and biolistic transformation.

As used herein, the term “transfection” refers to a process using anon-viral vector to deliver a gene to a target mammalian cell.

Typical transfection methods include electroporation, DNA biolistics,lipid-mediated transfection, compacted DNA-mediated transfection,liposomes, immunoliposomes, lipofectin, cationic agent-mediated,cationic facial amphiphiles (CFAs) (Nature Biotechnology 1996 14; 556),multivalent cations such as spermine, cationic lipids or polylysine, 1,2-bis (oleoyloxy)-3-(trimethylammonio) propane (DOTAP)-cholesterolcomplexes (Wolff and Trubetskoy 1998 Nature Biotechnology 16: 421) andcombinations thereof.

Uptake of naked nucleic acid constructs by mammalian cells is enhancedby several known transfection techniques for example those including theuse of transfection agents. Example of these agents include cationicagents (for example calcium phosphate and DEAE-dextran) and lipofectants(for example lipofectam™ and transfectam™). Typically, nucleic acidconstructs are mixed with the transfection agent to produce acomposition.

Viral Vectors

Alternatively, the vectors comprising nucleotide sequences of thepresent invention may be introduced into suitable host cells using avariety of viral techniques which are known in the art, such as forexample infection with recombinant viral vectors such as retroviruses,herpes simplex viruses and adenoviruses.

Preferably the vector is a recombinant viral vectors. Suitablerecombinant viral vectors include but are not limited to adenovirusvectors, adeno-associated viral (AAV) vectors, herpes-virus vectors, aretroviral vector, lentiviral vectors, baculoviral vectors, pox viralvectors or parvovirus vectors (see Kestler et al 1999 Human Gene Ther10(10):1619-32). In the case of viral vectors, gene delivery is mediatedby viral infection of a target cell.

The term “vector system” when applied to viral vector includes a vectorparticle capable of infecting a mammalian cell. There is also providedkits for the production of the vector particle, the constituents ofwhich will depend on the viral vector type on which the system is based.For example, a kit for a retrovirus may comprise:

-   -   i) a viral genome comprising a 5T4 nucleotide; and either    -   ii) one or more producer plasmids and a host cell, or    -   iii) a producer cell.        Retroviral Vectors

Examples of retroviruses include but are not limited to: murine leukemiavirus (MLV), human immunodeficiency virus (HIV), equine infectiousanaemia virus (EIAV), mouse mammary tumor virus (MMTV), Rous sarcomavirus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemiavirus (Mo-MLV), FBR murine osteosarcoma virus. (FBR MSV), Moloney murinesarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV); Avianmyelocytomatosis virus-29 (MC29), and Avian erythroblastosis virus(AEV).

Preferred vectors for use in accordance with the present invention arerecombinant viral vectors, in particular recombinant retroviral vectors(RRV) such as lentiviral vectors.

The term “recombinant retroviral vector” (RRV) refers to a vector withsufficient retroviral genetic information to allow packaging of an RNAgenome, in the presence of packaging components, into a viral particlecapable of infecting a target cell. Infection of the target cellincludes reverse transcription and integration into the target cellgenome. The RRV carries non-viral coding sequences which are to bedelivered by the vector to the target cell. An RRV is incapable ofindependent replication to produce infectious retroviral particleswithin the final target cell. Usually the RRV lacks a functional gag-poland/or env gene and/or other genes essential for replication. The vectorof the present invention may be configured as a split-intron vector. Asplit intron vector is described in PCT patent application WO 99/15683.

A detailed list of retroviruses may be found in Coffin etal(“Retroviruses” 1997 Cold Spring Harbour Laboratory Press Eds: J MCoffin, S M Hughes, H E Varmus pp 758-763).

Lentiviral Vectors

Lentiviruses can be divided into primate and non-primate groups.Examples of primate lentiviruses include but are not limited to: thehuman immunodeficiency virus (HIV), the causative agent of humanauto-immunodeficiency syndrome (AIDS), and the simian immunodeficiencyvirus (SIV). The non-primate lentiviral group includes the prototype“slow virus” visna/maedi virus (VMV), as well as the related caprinearthritis-encephalitis virus (CAEV), equine infectious anaemia virus(EIAV) and the more recently described feline immunodeficiency virus(FIV) and bovine immunodeficiency virus (BIV).

A distinction between the lentivirus family and other types ofretroviruses is that lentivirus s have the capability to infect bothdividing and non-dividing cells (Lewis et al1992 EMBO. J 11: 3053-3058;Lewis and Emerman 1994 J. Virol. 68: 510-516). In contrast, otherretroviruses—such as MLV—are unable to infect no-dividing cells such asthose that make up, for example, muscle, brain, lung and liver tissue.

Adenoviruses

In one embodiment of the present invention, the features of adenovirusesmay be combined with the genetic stability of retroviruses/lentiviruseswhich can be used to transduce target cells to become transientretroviral producer cells capable of stably infect neighbouring cells.Such retroviral producer cells which are engineered to express a 5T4poypeptide or 5T4-specific agent of the present invention can beimplanted in organisms such as animals or humans for use in thetreatment of disease such as cancer.

Pox Viruses

Preferred vectors for use in accordance with the present invention arerecombinant pox viral vectors such as fowl pox virus (FPV), entomopoxvirus, vaccinia virus such as NYVAC, canarypox virus, Modified vacciniaAnkara (MVA) or other non-replicating viral vector systems such as thosedescribed for example in WO 95/30018.

In a preferred embodiment the vector is MVA. General teachings on poxvectors and MVA can be found in WO 00/29428.

Expression of 5T4 proteins or antigens in recombinant pox viruses, suchas vaccinia viruses, requires the ligation of vaccinia promoters to thenucleic acid encoding 5T4. In order to do this a transfer plasmid isconstructed which contains at least one nucleic acid which codes for a5T4 antigen flanked by MVA DNA sequences. When this transfer plasmid isintroduced into cells infected with MVA, homologous recombination occurscausing the 5T4 nucleotide to be inserted into the MVA virus (Mackett etal 1982 PNAS 79: 7415-7419).

The transfer plasmid contains sequences flanking the left and the rightside of a naturally occurring deletion, e.g. deletion II, within the MVAgenome (Altenburger, W., Suter, C. P. and Altenburger J. (1989) Arch.Virol. 0.105, 15-27). The foreign DNA sequence is inserted between thesequences flanking the naturally occurring deletion.

For the expression of at least one nucleic acid, it is necessary forregulatory sequences, which are required for the transcription of thenucleic acid to be present upstream of the nucleic acid. Such regulatorysequences are known to those skilled in the art, and includes forexample those of the vaccinia 11 kDa gene as are described inEP-A-198,328, and those of the 7.5 kDa gene (EP-A-110,385).

The construct can be introduced into the MVA infected cells bytransfection, for example by means of calcium phosphate precipitation(Graham et al Virol. 52, 456-467-1973; Wigler et al Cell 777-785 [1979]by means of electroporation (Neumann et al EMBO J. 1, 841-845 [1982]),by microinjection (Graessmann et al Meth. Enzymology 101, 482-492(1983)), by means of liposomes (Straubinger et al Methods in Enzymology101, 512-527 (1983)), by means of spheroplasts (Schaffner, Proc. Natl.Acad. Sci. USA 77, 2163-2167 (1980)) or by other methods known to thoseskilled in the art. Transfection by means of liposomes is preferred.

Once the construct has been introduced into the eukaryotic cell and the5T4 antigen DNA has recombined with the viral DNA, the desiredrecombinant vaccinia virus, can be isolated, preferably with the aid ofa marker (Nakano et al Proc. Natl. Acad. Sci. USA. 79, 1593-1596 [1982],Franke et al Mol. Cell. Biol. 1918-1924 [1985], Chakrabarti et al Mol.Cell. Biol. 3403-3409 [1985], Fathi et al Virology 97-105 [1986]).

The present invention thus also provides a kit which comprises

-   -   a transfer plasmid which comprises a polynucleotide encoding a        5T4 antigen flanked by MVA DNA sequences.

The kit may also include a cell infected with MVA, such as BHK-21 or CEFcells, or wild-type MVA stock for infection purposes.

The nucleic acid must be inserted into a region (insertion region) inthe virus which does not affect virus viability of the resultantrecombinant virus. Such regions can be readily identified in a virus by,for example, randomly testing segments of virus DNA for regions thatallow recombinant formation without seriously affecting virus viabilityof the recombinant. One region that can readily be used and is presentin many viruses is the thymidine kinase (TK) gene. For example, the TKgene has been found in all pox virus genomes examined [leporipoxvirus:Upton, et al J. Virology 60:920 (1986) (shope fibroma virus);capripoxvirus: Gershon, et al J. Gen. Virol. 70:525 (1989) (Kenyasheep-1); orthopoxvirus: Weir, et al J. Virol 46:530 (1983) (vaccinia);Esposito, et al Virology 135:561 (1984) (monkeypox and variola virus);Hruby, et al PNAS, 80:3411 (1983) (vaccinia); Kilpatrick, et al Virology143:399 (1985) (Yaba monkey tumor virus); avipoxvirus: Binns, et al J.Gen. Virol 69:1275 (1988) (fowlpox); Boyle, et al Virology 156:355(1987) (fowlpox); Schnitzlein, et al J. Virological Method, 20:341(1988) (fowlpox, quailpox); entomopox (Lytvyn, et al J. Gen. Virol73:3235-3240 (1992)].

A promoter can be selected depending on the host and the target c IItype. For example in poxviruses, pox viral promoters should be used,such as the vaccinia 7.5K, or 40K or fowlpox C1. Artificial constructscontaining appropriate pox sequences can also be used. Enhancer elementscan also be used in combination to increase the level of expression.Furthermore, the use of inducible promoters, which are also well knownin the art, are preferred in some embodiments. A particularly preferredpromoter is a modified H5 promoter (Wyatt et al (1996) Vaccine (4)1451-1458).

Hybrid Viral Vectors

In a further broad aspect, the present invention provides a hybrid viralvector system for in vivo delivery of a nucleotide sequence encoding a5T4 poypeptide or 5T4-specific agent of the present invention, whichsystem comprises one or more primary viral vectors which encode asecondary viral vector, the primary vector or vectors capable ofinfecting a first target cell and of expressing therein the secondaryviral vector, which secondary vector is capable of transducing asecondary target cell.

Preferably the primary vector is obtainable from or is based on anadenoviral vector and/or the secondary viral vector is obtainable fromor is based on a retroviral vector preferably a lentiviral vector.

Targeted Vector

The term “targeted vector” refers to a vector whose ability toinfect/transfect/transduce a cell or to be expressed in a host and/ortarget cell is restricted to certain cell types within the hostorganism, usually cells having a common or similar phenotype.

Replication Vectors

The nucleotide sequences encoding the 5T4 poypeptide or 5T4-specificagent of the present invention may be incorporated into a recombinantreplicable vector. The vector may be used to replicate the nucleotidesequence in a compatible host cell. Thus in one embodiment of thepresent invention, the invention provides a method of making the 5T4poypeptide or 5T4-specific agent of the present invention by introducinga nucleotide sequence of the present invention into a replicable vector,introducing the vector into a compatible host cell, and growing the hostcell under conditions which bring about replication of the vector. Thevector may be recovered from the host cell.

Expression Vector

Preferably, a nucleotide sequence of present invention which is insertedinto a vector is operably linked to a control sequence that is capableof providing for the expression of the coding sequence, such as thecoding sequence of the 5T4 poypeptide or 5T4-specific agent of thepresent invention by the host cell, i.e. the vector is an expressionvector. The 5T4 poypeptide or 5T4-specific agent produced by a hostrecombinant cell may be secreted or may be contained intracellularlydepending on the sequence and/or the vector used. As will be understoodby those of skill in the art, expression vectors containing the 5T4poypeptide or 5T4-specific agent coding sequences can be designed withsignal sequences which direct secretion of the 5T4 poypeptide or5T4-specific agent coding sequences through a particular prokaryotic oreukaryotic cell membrane.

Expression in Vitro

The vectors of the present invention may be transformed or transfectedinto a suitable host cell and/or a target cell as described below toprovide for expression of a 5T4 poypeptide or 5T4-specific agent of thepresent invention. This process may comprise culturing a host celland/or target cell transformed with an expression vector underconditions to provide for expression by the vector of a coding sequenceencoding the 5T4 poypeptide or 5T4-specific agent and optionallyrecovering the expressed 5T4 poypeptide or 5T4-specific agent. Thevectors may be for example, plasmid or virus vectors provided with anorigin of replication, optionally a promoter for the expression of thesaid polynucleotide and optionally a regulator of the promoter. Thevectors may contain one or more selectable marker genes, for example, anampicillin resistance gene in the case of a bacterial plasmid or aneomycin resistance gene for a mammalian vector. The expression of the5T4 poypeptide or 5T4-specific agent of the invention may beconstitutive such that they are continually produced, or inducible,requiring a stimulus to initiate expression. In the case of inducibleexpression, 5T4 poypeptide or 5T4-specific agent production can beinitiated when require by, for example, addition of an inducer substanceto the culture medium, for example dexamethasone or IPTG.

Host/Target Cells

Host and/or target cells comprising nucleotide sequences of the presentinvention may be used to express the 5T4 polypeptide or 5T4-specificagents of the present invention under in vitro, in vivo and ex vivoconditions.

The term “host cell and/or target cell” includes any cell derivable froma suitable organism which a vector is capable of transfecting ortransducing. Examples of host and/or target cells can include but arenot limited to cells capable of expressing the 5T4 polypeptide or5T4-specific agent of the present invention under in vitro, in vivo andex vivo conditions. Examples of such cells include but are not limitedto macrophages, endothelial cells or combinations thereof. Furtherexamples include respiratory airway epithelial cells, hepatocytes,muscle cells, cardiac myocytes, synoviocytes, primary mammary epithelialcess and post-mitotically terminally differentiated non-replicatingcells such as macrophages and/or neurons.

In a preferred embodiment, the cell is a mammalian cell.

In a highly preferred embodiment, the cell is a canine or feline cell.

The term “organism” includes any suitable organism. In a preferredembodiment, the organism is a mammal. In a highly preferred embodiment,the organism is a dog or cat.

Although the 5T4 polypeptide or 5T4-specific agent of the invention maybe produced using prokaryotic cells as host cells, it is preferred touse eukaryotic cells, for example yeast, insect or mammalian cells, inparticular mammalian cells. Suitable host cells include bacteria such asE. coli, yeast, mammalian cell lines and other eukaryotic cell lines,for example insect Sf9 cells.

The present invention also provides a method comprising transforming ahost and/or target cell with a or the nucleotide sequence(s) of thepresent invention.

The term “transformed cell” means a host cell and/or a target cellhaving a modified genetic structure. With the present invention, a cellhas a modified genetic structure when a vector according to the presentinvention has been introduced into the cell.

Host cells and/or a target cells may be cultured under suitableconditions which allow expression of the 5T4 polypeptide or 5T4-specificagent of the invention.

The present invention also provides a method comprising culturing atransformed host cell—which cell has been transformed with a or thenucleotide sequence(s) according to the present invention underconditions suitable for the expression of the 5T4 polypeptide or5T4-specific agent encoded by said nucleotide sequence(s).

The present invention also provides a method comprising culturing atransformed host cell—which cell has been transformed with a or thenucleotide sequence(s) according to the present invention or aderivative, homologue, variant or fragment thereof—under conditionssuitable for the expression of the 5T4 polypeptide or 5T4-specific agentencoded by said nucleotide sequence(s); and then recovering said 5T4polypeptide or 5T4-specific agent from the transformed host cellculture.

The 5T4 polypeptide or 5T4-specific agent of the present invention canbe extracted from host cells by a variety of techniques known in theart, including enzymatic, chemical and/or osmotic lysis and physicaldisruption. The 5T4 polypeptide or 5T4-specific agent may be purifiedand isolated in a manner known per se.

Regulation of Expression In Vitro/Vivo/Ex Vivo

The present invention also encompasses gene therapy whereby the 5T4polypeptide or 5T4-specific agent encoding nucleotide sequence(s) of thepresent invention is regulated in vitrolin vivolex vivo. For example,expression regulation may be accomplished by administering compoundsthat bind to the 5T4 polypeptide or 5T4-specific agent encodingnucleotide sequence(s) of the present invention, or control regionsassociated with the 5T4 polypeptide or 5T4-specific agent encodingnucleotide sequence of the present invention, or its corresponding RNAtranscript to modify the rate of transcription or translation.

Control Sequences

Control sequences operably linked to sequences encoding the 5T4polypeptide or 5T4-specific agent of the present invention includepromoters/enhancers and other expression regulation signals. Thesecontrol sequences may be selected to be compatible with the host celland/or target cell in which the expression vector is designed to beused. The control sequences may be modified, for example by the additionof further transcriptional regulatory elements to make the level oftranscription directed by the control sequences more responsive totranscriptional modulators.

Operably Linked

The term “operably linked” means that the components described are in arelationship permitting them to function in their intended manner. Aregulatory sequence “operably linked” to a coding sequence is ligated insuch a way that expression of the coding sequence is achieved undercondition compatible with the control sequences.

Preferably the nucleotide sequence of the present invention is operablylinked to a transcription unit.

The term “transcription unit(s)” as described herein are regions ofnucleic acid containing coding sequences and the signals for achievingexpression of those coding sequences independently of any other codingsequences. Thus, each transcription unit generally comprises at least apromoter, an optional enhancer and a polyadenylation signal.

Promoters

The term promoter is well-known in the art and is used to indicate atranscription factor (complex) binding site. The term encompassesnucleic acid regions ranging in size and complexity from minimalpromoters to promoters including upstream elements and enhancers.

The promoter is typically selected from promoters which are functionalin mammalian, cells, although prokaryotic promoters and promotersfunctional in other eukaryotic cells may be used. The promoter istypically derived from promoter sequences of viral or eukaryotic genes.For example, it may be a promoter derived from the genome of a cell inwhich expression is to occur. With respect to eukaryotic promoters, theymay be promoters that function in a ubiquitous manner (such as promotersof α-actin, β-actin, tubulin) or, alternatively, a tissue-specificmanner (such as promoters of the genes for pyruvate kinase).

Hypoxic Promoters/Enhancers

The enhancer and/or promoter may be preferentially active in a hypoxicor ischaemic or low glucose environment, such that the 5T4 polypeptideor 5T4-specific agent encoding nucleotide sequence(s) is preferentiallyexpressed in the particular tissues of interest, such as in theenvironment of a tumor, arthritic joint or other sites of ischaemia.Thus, any significant biological effect or deleterious effect of the 5T4polypeptide-or 5T4-specific agent encoding nucleotide sequence(s) on theindividual being treated may be reduced or eliminated. The enhancerelement or other elements conferring regulated expression may be presentin multiple copies. Likewise, or in addition, the enhancer and/orpromoter may be preferentially active in one or more specific celltypes—such as any one or more of macrophages, endothelial cells orcombinations thereof. Further examples may include but are not limitedto respiratory airway epithelial cells, hepatocytes, muscle cells,cardiac myocytes, synoviocytes, primary mammary epithelial cells andpost-mitotically terminally differentiated non-replicating cells such asmacrophages and/or neurons.

Tissue-Specific Promoters

The promoters of the present invention may be tissue-specific promoters.Examples of suitable tissue restricted promoters/enhancers are thosewhich are highly active in tumor cells such as a promoter/enhancer froma MUC1 gene, a CEA gene or a 5T4 antigen gene. Examples of temporallyrestricted promoters/enhancers are those which are responsive toischaemia and/or hypoxia, such as hypoxia response elements or thepromoter/enhancer of a grp78 or a grp94 gene. The alpha fetoprotein(AFP) promoter is also a tumor-specific promoter. One preferredpromoter-enhancer combination is a human cytomegalovirus (hCMV) majorimmediate early (MIE) promoter/enhancer combination.

Preferably the promoters of the present invention are tissue specific.That is, they are capable of driving transcription of a 5T4 polypeptideor 5T4-specific agent encoding nucleotide sequence(s) in one tissuewhile remaining largely “silent” in other tissue types.

The term “tissue specific” means a promoter which is not restricted inactivity to a single tissue type but which nevertheless showsselectivity in that they may be active in one group of tissues and lessactive or silent in another group. A desirable characteristic of thepromoters of the present invention is that they posess a relatively lowactivity in the absence of activated hypoxia-regulated enhancerelements, even in the target tissue. One means of achieving this is touse “silencer” elements which suppress the activity of a selectedpromoter in the absence of hypoxia.

The term “hypoxia” means a condition under which a particular organ ortissue receives an inadequate supply of oxygen.

The level of expression of a or the 5T4 polypeptide or 5T4-specificagent encoding nucleotide sequence(s) under the control of a particularpromoter may be modulated by manipulating the promoter region. Forexample, different domains within a promoter region may possessdifferent gene regulatory activities. The roles of these differentregions are typically assessed using vector constructs having differentvariants of the promoter with specific regions deleted (that is,deletion analysis). This approach may be used to identify, for example,the smallest region capable of conferring tissue specificity or thesmallest region conferring hypoxia sensitivity.

A number of tissue specific promoters, described above, may beparticularly advantageous in practising the present invention. In mostinstances, these promoters may be isolated as convenient restrictiondigestion fragments suitable for cloning in a selected vector.Alternatively, promoter fragments may be isolated using the polymerasechain reaction. Cloning of the amplified fragments may be facilitated byincorporating restriction sites at the 5′ end of the primers.

Inducible Promoters

The promoters of the present invention may also be promoters thatrespond to specific stimuli, for example promoters that bind steroidhormone receptors. Viral promoters may also be used, for example theMoloney murine leukaemia virus long terminal repeat (MMLV LTR) promoter,the rous sarcoma virus (RSV) LTR promoter or the human cytomegalovirus(CMV) IE promoter.

It may also be advantageous for the promoters to be inducible so thatthe levels of expression of the heterologous gene can be regulatedduring the life-time of the cell. Inducible means that the levels ofexpression obtained using the promoter can be regulated.

Enhancer

In addition, any of these promoters may be modified by the addition offurther regulatory sequences, for example enhancer sequences. Chimericpromoters may also be used comprising sequence elements from two or moredifferent promoters described above.

The term “enhancer” includes a DNA sequence which binds to other proteincomponents of the transcription initiation complex and thus facilitatesthe initiation of transcription directed by its associated promoter.

The in vitro/in vivo/ex vivo expression of the 5T4 polypeptide or5T4-specific agent of the present invention may be used in combinationwith a protein of interest (POI) or a nucleotide sequence of interest(NOI) encoding same.

Combination With POIs/NOIs

The 5T4 polypeptide or 5T4-specific agent of the present invention ornucleotide sequence encoding same may be used in combination with a POI,such as a pro-drug activating enzyme either directly or by vectordelivery to, for example, a target cell or target tissue. Instead of oras well as being selectively expressed in target tissues, the 5T4polypeptide or 5T4-specific agent of the present invention or nucleotidesequence encoding same may be used in combination with another POI suchas a pro-drug activation enzyme or enzymes or with a nucleotidesequences of interest (NOI) or NOIs which encode a pro-drug activationenzyme or enzymes. These pro-drug activation enzyme or enzymes may haveno significant effect or no deleterious effect until the individual istreated with one or more pro-drugs upon which the appropriate pro-drugenzyme or enzymes act. In the presence of the active POI or NOI encodingsame, treatment of an individual with the appropriate pro-drug may leadto enhanced reduction in the disease condition such as a reduction intumor growth or survival.

Pro-Drug POIs

A POI, such as a pro-drug activating enzyme, may be delivered to adisease site, such as a tumor site for the treatment of a cancer. Ineach case, a suitable pro-drug is used in the treatment of the patientin combination with the appropriate pro-drug activating enzyme. Anappropriate pro-drug may be administered in conjunction with the 5T4polypeptide or 5T4-specific agent or vector comprising the nucleotidesequence encoding same. Examples of pro-drugs include: etoposidephosphate (with alkaline phosphatase, Senter et al1988 Proc Natl AcadSci 85: 4842-4846); 5-fluorocytosine (with cytosine deaminase, Mullen etal1994 Cancer Res 54: 1503-1506);Doxorubicin-N-p-hydroxyphenoxyacetamide (with Penicillin-V-Amidase, Kerret al1990 Cancer Immunol Immunother 31: 202-206);Para-N-bis(2-chloroethyl) aminobenzoyl glutamate (with carboxypeptidaseG2); Cephalosporin nitrogen mustard carbamates (with βb-lactamase);SR4233 (with P450 Reductase); Ganciclovir (with HSV thymidine kinase,Borrelli et all 988 Proc Natl Acad Sci 85: 7572-7576); mustard pro-drugswith nitroreductase (Friedlos et al1997 J Med. Chem 40: 1270-1275) andCyclophosphamide (with P450 Chen et al1996 Cancer Res 56: 1331-1340).

Examples of suitable pro-drug activation enzymes for use in theinvention include a thymidine phosphorylase which activates the5-fluoro-uracil pro-drugs capcetabine and furtulon; thymidine kinasefrom Herpes Simplex Virus which activates ganciclovir; a cytochrome P450which activates a pro-drug such as cyclophosphamide to a DNA damagingagent; and cytosine deaminase which activates 5-fluorocytosine.Preferably, a pro-drug activating enzyme of human origin is used.

POIs and NOIs

Other suitable proteins of interest (POIs) or NOIs encoding same for usein the present invention include those that are of therapeutic and/ordiagnostic application such as, but are not limited to: sequencesencoding cytokines, chemokines, hormones, antibodies, engineeredimmunoglobulin-like molecules, a single chain antibody, fusion proteins,enzymes, immune co-stimulatory molecules, immunomodulatory molecules,anti-sense RNA, a transdominant negative mutant of a target protein, atoxin, a conditional toxin, an antigen, a tumor suppressor protein andgrowth factors, membrane proteins, vasoactive proteins and peptides,anti-viral proteins and ribozymes, and derivatives thereof (such as withan associated reporter group). When included, the POIs or NOIs encodingsame may be typically operatively linked to a suitable promoter, whichmay be a promoter driving expression of a ribozyme(s), or a differentpromoter or promoters, such as in one or more specific cell types.

Bystander Effect

The POI and/or NOI encoding same may be proteins which are secreted froma cell. Alternatively the POI expression products are not secreted andare active within the cell. In either event, it is preferred for the POIexpression product to demonstrate a bystander effector or a distantbystander effect; that is the production of the expression product inone cell leading to the killing of additional, related cells, eitherneighbouring or distant (e.g. metastatic), which possess a commonphenotype.

Suitable POIs or NOIs encoding same for use in the present invention inthe treatment or prophylaxis of cancer include proteins which: destroythe target cell (for example a ribosomal toxin), act as: tumorsuppressors (such as wild-type p53); activators of anti-tumor immunemechanisms (such as cytokines, co-stimulatory molecules andimmunoglobulins); inhibitors of angiogenesis; or which provide enhanceddrug sensitivity (such as pro-drug activation enzymes); indirectlystimulate destruction of target cell by natural effector cells (forexample, strong antigen to stimulate the immune system or convert aprecursor substance to a toxic substance which destroys the target cell(for example a prodrug activating enzyme). Encoded proteins could alsodestroy bystander tumor cells (for example with secreted antitumorantibody-ribosomal toxin fusion protein), indirectly stimulatedestruction of bystander tumor cells (for example cytokines to stimulatethe immune system or procoagulant proteins causing local vascularocclusion) or convert a precursor substance to a toxic substance whichdestroys bystander tumor cells (eg an enzyme which activates a prodrugto a diffusible drug).

Also, the delivery of NOI(s) encoding antisense transcripts or ribozymeswhich interfere with expression of cellular genes for tumor persistence(for example against aberrant myc transcripts in Burkitts lymphoma oragainst bcr-abl transcripts in chronic myeloid leukemia. The use ofcombinations of such POIs and/or NOIs encoding same is also envisaged.

Examples of hypoxia regulatable therapeutic NOIs can be found inPCT/GB95/00322 (WO-A9521927).

Coupling

The 5T4 polypeptide or 5T4-specific agent of the present invention canbe coupled to other molecules using standard methods. The amino andcarboxyl termini of 5T4 polypeptide or 5T4-specific agent may beisotopically and nonisotopically labeled with many techniques, forexample radiolabeling using conventional techniques (tyrosineresidues-chloramine T, iodogen, lactoperoxidase; lysineresidues-Bolton-Hunter reagent). These coupling techniques are wellknown to those skilled in the art. The coupling technique is chosen onthe basis of the functional groups available on the amino acidsincluding, but not limited to amino, sulfhydral, carboxyl, amide,phenol, and imidazole. Various reagents used to effect these couplingsinclude among others, glutaraldehyde, diazotized benzidine,carbodiimide, and p-benzoquinone.

Chemical Coupling

The 5T4 polypeptide or 5T4-specific agent of the present invention maybe chemically coupled to isotopes, enzymes, carrier proteins, cytotoxicagents, fluorescent molecules, radioactive nucleotides and othercompounds for a variety of applications including but not limited toimaging/prognosis, diagnosis and/or therapy. The efficiency of thecoupling reaction is determined using different techniques appropriatefor the specific r action. For example, radiolabeling of an 5T4polypeptide or 5T4-specific agent peptide with ¹²⁵I is accomplishedusing chloramine T and Na¹²⁵I of high specific activity. The reaction isterminated with sodium metabisulfite and the mixture is desalted ondisposable columns. The labeled peptide is eluted from the column andfractions are collected. Aliquots are removed from each fraction andradioactivity measured in a gamma counter. In this manner, the unreactedNa ¹²⁵ I is separated from the labeled 5T4 polypeptide or 5T4-specificagent. The peptide fractions with the highest specific radioactivity arestored for subsequent use such as analysis of the ability to bind to a5T4 polypeptide or 5T4-specific agent.

Imaging

The use of labelled 5T4-specific agents of the present invention withshort lived isotopes enables visualization quantitation of 5T4 in vivousing autoradiographic, or modem radiographic or other membrane bindingtechniques such as positron emission tomography in order to locatetumors with 5T4. This application provides important diagnostic and/orprognostic research tools.

Conjugates

In other embodiments, the 5T4 polypeptide or 5T4-specific agent of theinvention is coupled to a scintigraphic radiolabel, a cytotoxic compoundor radioisotope, an enzyme for converting a non-toxic prodrug into acytotoxic drug, a compound for activating the immune system in order totarget the resulting conjugate to a disease site such as a colon tumor,or a cell-stimulating compound. Such conjugates have a “bindingportion”, which consists of the 5T4 polypeptide or 5T4-specific agent ofthe invention, and a “functional portion”, which consists of theradiolabel, toxin or enzyme. Different 5T4 polypeptide or 5T4-specificagents can be synthesized for use in several applications including butnot limited to the linkage of a 5T4 polypeptide or 5T4-specific agent tocytotoxic agents for targeted killing of cells that bind the 5T4polypeptide or 5T4-specific agent.

The binding portion and the functional portion of the conjugate (if alsoa peptide or poypeptide) may be linked together by any of theconventional ways of cross linking polypeptides, such as those generallydescribed in O'Sullivan et al(Anal. Biochem 1979: 100, 100-108). Forexample, one portion may be enriched with thiol groups and the otherportion reacted with a bifunctional agent capable of reacting with thosethiol groups, for example the N-hydroxysuccinimide ester of iodoaceticacid (NHIA) or N-succinimidyl-3-(2-pyridyidithio)propionate (SPDP).Amide and thioether bonds, for example achieved withm-maleimidobenzoyl-N-hydroxysuccinimide ester, are generally more stablein vivo than disulphide bonds.

Alternatively, if the binding portion contains carbohydrates, such aswould be the case for an antibody or some antibody fragments, thefunctional portion may be linked via the carbohydrate portion using thelinking technology in EP 0 088 695.

The functional portion of the conjugate may be an enzyme for convertinga non-toxic prodrug into a toxic drug, for example the conjugates ofBagshawe and his colleagues (Bagshawe (1987) Br. J. Cancer 56, 531;Bagshawe et al(Br. J. Cancer 1988: 58, 700); WO 88/07378) orcyanide-releasing systems (WO 91/11201).

The conjugate may be purified by size exclusion or affinitychromatography, and tested for dual biological activities. The antigenimmunoreactivity may be measured using an enzyme-linked immunosorbentassay (ELISA) with immobilised antigen and in a live cellradioimmunoassay. An enzyme assay may be used for β-glucosidase using asubstrate which changes in absorbance when the glucose residues arehydrolysed, such as oNPG (o-nitrophenyl-βD-glucopyranoside), liberating2-nitrophenol which is measured spectrophotometrically at 405 nm.

The stability of the conjugate may be tested in vitro initially byincubating at 37° C. in serum, followed by size exclusion FPLC analysis.Stability in vivo can be tested in the same way in mice by analysing theserum at various times after injection of the conjugate. In addition, itis possible to radiolabel the 5T4 polypeptide or 5T4-specific agent with¹²⁵I, and the enzyme with ¹³¹I before conjugation, and to determine thebiodistribution of the conjugate, free 5T4 polypeptide or 5T4-specificagent and free enzyme in mice.

Alternatively, the conjugate may be produced as a fusion compound byrecombinant DNA techniques whereby a length of DNA comprises respectiveregions encoding the two portions of the conjugate either adjacent toone another or separated by a region encoding a linker peptide whichdoes not destroy the desired properties of the conjugate.

Conceivably, two of the functional portions of the compound may overlapwholly or partly. The DNA is then expressed in a suitable host in knownways.

Diagnostic Kits

The present invention also includes diagnostic methods and kits fordetection and measurement of 5T4 in biological fluids and tissues, andfor localization of 5T4 in tissues. The 5T4 polypeptide or 5T4-specificagent of thepresent invention that possess high binding specificity canbe used to establish easy to use kits for rapid, reliable, sensitive,and specific measurement and localization of a 5T4 in extracts ofplasma, urine, tissues, and in cell culture media. The 5T4 polypeptideor 5T4-specific agent of the present invention may also be used in adiagnostic method and kit to permit detection of circulating 5T4 which,in certain situations, may indicate the progression of a disease statesuch as the spread of micrometastases by primary tumors in situ.

These kits may include but are not limited to the following techniques;competitive and non-competitive assays, radioimmunoassay,bioluminescence and chemiluminescence assays, fluorometric assays,sandwich assays, immunoradiometric assays, dot blots, enzyme linkedassays including ELISA, microtiter plates, antibody coated strips ordipsticks for rapid monitoring of urine or blood, andimmunocytochemistry. For each kit the range, sensitivity, precision,reliability, specificity and reproducibility of the assay areestablished. Intraassay and interassay variation is established at 20%,50% and 80% points on the standard curves of displacement or activity.

One example of an assay kit commonly used in research and in the clinicis a radioimmunoassay (RIA) kit. After successful radioiodination andpurification of a 0.5T4-specific agent, the antiserum possessing thehighest titer is added at several dilutions to tubes containing arelatively constant amount of radioactivity, such as 10,000 cpm, in asuitable buffer system. Other tubes contain buffer or preimmune serum todetermine the non-specific binding. After incubation at 4° C. for 24hours, protein A is added and the tubes are vortexed, incubated at roomtemperature for 90 minutes, and centrifuged at approximately 2000-2500times g at 4° C. to precipitate the complexes of antiserum bound to thelabeled 5T4-specific antibody. The supernatant is removed by aspirationand the radioactivity in the pellets counted in a gamma counter. Theantiserum dilution that binds approximately 1.0 to 40% of the labeled5T4-specific agent after subtraction of the non-specific binding isfurther characterized.

Immunohistochemistry

An immunohistochemistry kit may also be used for localization of 5T4 intissues and cells. This immunohistochemistry kit provides instructions,a 5T4-specific antibody, and possibly blocking serum and secondaryantiserum linked to a fluorescent molecule such as fluoresceinisothiocyanate, or to some other reagent used to visualize the primaryantiserum. Immunohistochemistry techniques are well known to thoseskilled in the art. This immunohistochemistry kit permits localizationof 5T4 in tissue sections and cultured cells using both light andelectron microscopy. It is used for both research and clinical purposes.For example, tumors are biopsied or collected and tissue sections cutwith a microtome to examine sites of 5T4 production. Such information isuseful for diagnostic and possibly therapeutic purposes in the detectionand treatment of diseases such as cancer.

Foetal Cell Analysis

The 5T4 polypeptide and 5T4-specific agents of the present invention arealso useful in methods for isolating foetal cells from maternal blood.Isolation of foetal cells from maternal blood has been proposed as anon-invasive alternative to aminocentesis (see WO 97/30354).

5T4 is known to be expressed at very high levels on trophoblasts. Thusan antibody against 5T4 may be used to isolate trophoblasts frommaternal blood.

Thus the present invention also provides a method for isolating a foetalcell from maternal blood using an 5T4-specific agent Of the presentinvention

The foetal cell may, for example, be a trophoblast or an erythrocyte.

The maternal/foetal cells are preferably from a cat or a dog, such thatthe isolation method is part of a veterinary application.

The isolation process may form part of a diagnostic method. For example,the foetal cells may then be subject to biochemical or genetic sampling.Such a procedure sould be used to test for foetal abnormalities such asDowns syndrome, or to determine the sex of the foetus(es).

Combination Therapy

The 5T4 polypeptide or 5T4-specific agents of the present invention maybe used in combination with other compositions and procedures for thetreatment of diseases. By way of example, the 5T4 polypeptide or5T4-specific agents may also be used in combination with conventionaltreatments of diseases such as cancer. By ways of further example, atumor may be treated conventionally with surgery, radiation orchemotherapy combined with a 5T4 polypeptide or 5T4-specific agent or a5T4 polypeptide or 5T4-specific agent may be subsequently administeredto the patient to extend the dormancy of micrometastases and tostabilize any residual primary tumor.

Delivery

The 5T4 polypeptide or 5T4-specific agent can be delivered with anothertherapeutically effective agent at the same moment in time and at thesame site. Alternatively, the 5T4 polypeptide or 5T4-specific agent andthe therapeutically effective agent may be delivered at a different timeand to a different site. The 5T4 polypeptide or 5T4-specific agent andthe therapeutically effective agent may even be delivered in the samedelivery vehicle for the prevention and/or treatment of a diseasecondition such as cancer.

Therapeutic strategies based on the use of the 5T4-specific agentinclude the recruitment and activation of T cells by using a fusion a5T4-specific agent fragment with the bacterial superantigenstaphylococcal enterotoxin (Dohlsten et al1994) or by using bispecificantibodies, directed to both 5T4 and the T-cell CD3 antigen (Kroesen etal 1994). Anti-5T4 antibodies may also be conjugated to differentbacterial toxins to yield potent immunotoxins (LeMaistre et al 1987;Zimmermann et al 1997).

5T4 polypeptide or 5T4-specific agents may be used in combination withcytotoxic agents for the prevention and/or treatment of disease statessuch as angiogenesis and/or cancer. Cytotoxic agents such as ricin,linked to a 5T4-specific agent may provide a tool for the destruction ofcells expressing 5T4. These cells may be found in many locations,including but not limited to, micrometastases and primary tumors.

Dosage

The dosage of the composition of the present invention will depend onthe disease state or condition being treated and other clinical factorssuch as weight and condition of the human or animal and the route ofadministration of the compound. Depending upon the half-life of theactive agent in the particular animal or human, the agent (e.g. anti-5T4antibody) can be administered between several times per day to once aweek. It is to be understood that the present invention has applicationprimarily for veterinary use. The methods of the present inventioncontemplate single as well as multiple administrations, given eithersimultaneously or over an extended period of time.

Formulations

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example, sealed ampoules and vials, and may be stored ina freeze-dried (lyophilized) condition requiring only the addition ofthe sterile liquid carrier, for example, water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

The compositions of the present invention may be effective in preventingand/or treating diseases such as cancer related diseases. The presentinvention includes the method of treating diseases such as cancerrelated disease with an effective amount of a composition of the presentinvention. The 5T4 polypeptide or 5T4-specific agent of the presentinvention can be provided as a synthetic peptide or an isolated andsubstantially purified proteins or protein fragments or a combinationthereof in pharmaceutically acceptable compositions using formulationmethods known to those of ordinary skill in the art. These compositionscan be administered by standard routes. These include but are notlimited to: oral, rectal, ophthalmic (including intravitreal orintracameral), nasal, topical (including buccal and sublingual),intrauterine, vaginal or parenteral (including subcutaneous,intraperitoneal, intramuscular, intravenous, intradermal, intracranial,intratracheal, and epidural) transdermal, intraperitoneal, intracranial,intracerebroventricular, intracerebral, intravaginal, intrauterine, orparenteral (e.g., intravenous, intraspinal, subcutaneous orintramuscular) routes.

The compositions may conveniently be presented in unit dosage form andmay be prepared by conventional pharmaceutical techniques. Suchtechniques include the step of bringing into association the activeingredient and the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredient with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

In addition, the compositions of the present invention may beincorporated into biodegradable polymers allowing for sustained releaseof the compound, the polymers being implanted in the vicinity of wheredrug delivery is desired, for example, at the site of a tumor orimplanted so that the active agent is slowly released systemically. Thebiodegradable polymers and their use are described, for example, indetail in Brem et al(J. Neurosurg 1991 74:441-446). Osmotic minipumpsmay also be used to provide controlled delivery of high concentrationsof active agents through cannulae to the site of interest, such asdirectly into a metastatic growth or into the vascular supply to thattumor.

The 5T4 polypeptide or 5T4-specific agents of the present invention maybe linked to cytotoxic agents which are infused in a manner designed tomaximize delivery to the desired location. For example, ricin-linkedhigh affinity 5T4 polypeptide or 5T4-specific agents are deliveredthrough a cannula into vessels supplying the target site or directlyinto the target. Such agents are also delivered in a controlled mannerthrough osmotic pumps coupled to infusion cannulae.

Preferred unit dosage formulations are those containing a daily dose orunit, daily sub-dose, as herein above recited, or an appropriatefraction thereof, of the administered ingredient. It should beunderstood that in addition'to the ingredients, particularly mentionedabove, the formulations of the present invention may include otheragents conventional in the art having regard to the type of formulationin question.

The conjugates may be administered in any suitable way, usuallyparenterally, for example intravenously or intraperitoneally, instandard sterile, non-pyrogenic formulations of diluents and carriers,for example isotonic saline (when administered intravenously). Once thconjugate has bound to the target cells and been cleared from thebloodstream (if necessary), which typically takes a day or so, thepro-drug is administered, usually as a single infused dose, or the tumoris imaged. If needed, because the conjugate may be immunogenic,cyclosporin or some other immunosuppressant can be administered toprovide a longer period for treatment but usually this will not benecessary.

The timing between administrations of the conjugate and pro-drug may beoptimised in a routine way since disease/normal tissue ratios ofconjugate (at least following intravenous delivery) are highest afterabout 4-6 days, whereas at this time the absolute amount of conjugatebound to the 5T4, in terms of percent of injected dose per gram, islower than at earlier times.

Therefore, the optimum interval between administration of the conjugateand the pro-drug will be a compromise between peak concentration of theenzyme at the disease site and the best distribution ratio betweendisease and normal tissues. The dosage of the conjugate will be chosenby the physician according to the usual criteria. At least in the caseof methods employing a targeted enzyme such as β-glucosidase andintravenous amygdalin as the toxic pro-drug, 1 to 50 daily doses of 0.1to 10.0 grams per square metr of body surface area, preferably 1.0-5.0g/m² are likely to be appropriate. For oral therapy, three doses per dayof 0.05 to 10.0 g, preferably 1.0-5.0 g, for one to fifty days may beappropriate. The dosage of the conjugate will similarly be chosenaccording to normal criteria, particularly with reference to the type,stage and location of the disease tissue and the weight of the patient.The duration of treatment will depend in part upon the rapidity andextent of any immune reaction to the conjugate.

The functional portion of the conjugate, when used for diagnosis,usually comprises and may consist of a radioactive atom forscintigraphic studies, for example technetium 99 m (^(99m)Tc) oriodine-123 (¹²³I), or a spin label for nuclear magnetic resonance (nmr)imaging (also known as magnetic resonance imaging, mri), such asiodine-123 again, iodine-313, indium-111, fluorine-19, carbon-13,nitrogen-15, oxygen-17, gadolinium, manganese or iron.

When used in a compound for selective destruction of, for example, thetumor, the functional portion of the 5T4-specific agent may comprise ahighly radioactive atom, such as iodine-131, rhenium-186, rhenium-188,yttrium-90 or lead-212, which emits enough energy to destroyneighbouring cells, or a cytotoxic chemical compound such asmethotrexate, adriamicin, vinca alkaliods (vincristine, vinblastine,etoposide), daunorubicin or other intercalating agents.

The radio- or other labels may be incorporated in the 5T4-specific agentconjugate in known ways. For example, the peptide may be biosynthesisedor may be synthesised by chemical amino acid synthesis using suitableamino acid precursors involving, for example, fluorine-19 in place ofhydrogen. Labels such as ^(99m)Tc, ¹²³I, ¹⁸⁶Rh, ¹⁸⁸Rh and ¹¹¹In can beattached via a cysteine residue in the peptide. Yttrium-90 can beattached via a lysine residue. The IODOGEN method (Fraker et al(1978)Biochem. Biophys. Res. Commun. 80: 49-57 can be used to incorporateiodine-123. “Monoclonal Antibodies in Immunoscinigraphy” (Chatal, CRCPress 1989) describes other methods in detail.

Pharmaceutical Compositions

The third aspect of the invention refers to vaccines priming andboosting compositions, agents and kits. Any and all of the products ofthis aspect of the invention can be considered to be a pharmaceuticalcomposition.

The pharmaceutical compositions will typically be for animal usage inveterinary medicine and will typically comprise any one or more of apharmaceutically acceptable diluent, carrier, or excipient. Acceptablecarriers or diluents for therapeutic use are well known in thepharmaceutical art, and are described, for example, in Remington's.Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).The choice of pharmaceutical carrier, excipient or diluent can beselected with regard to the intended route of administration andstandard pharmaceutical practice. The pharmaceutical compositions maycomprise as—or in addition to—the carrier, excipient or diluent anysuitable binder(s), lubricant(s), suspending agent(s), coating agent(s),solubilising agent(s).

Preservatives, stabilizers, dyes and even flavouring agents may beprovided in the pharmaceutical composition. Examples of preservativesinclude sodium benzoate, sorbic acid and esters of p-hydroxybenzoicacid. Antioxidants and suspending agents may be also used.

There may be different composition/formulation requirements dependent onthe different delivery systems. By way of example, the pharmaceuticalcomposition of the present invention may be formulated to be deliveredusing a mini-pump or by a mucosal route, for example, as a nasal sprayor aerosol for inhalation or ingestable solution, or parenterally inwhich the composition is formulated by an injectable form, for delivery,by, for example, an intravenous, intramuscular or subcutaneous route.Alternatively, the formulation may be designed to be delivered by bothroutes.

Where the pharmaceutical composition is to be delivered mucosallythrough the gastrointestinal mucosa, it should be able to remain stableduring transit though the gastrointestinal tract; for example, it shouldbe resistant to proteolytic degradation, stable at acid pH and resistantto the detergent effects of bile.

Where appropriate, the pharmaceutical compositions can be administeredby inhalation, in the form of a suppository or pessary, topically in theform of a lotion, solution, cream, ointment or dusting powder, by use ofa skin patch, orally in the form of tablets containing excipients suchas starch or lactose or chalk, or in capsules or ovules either alone orin admixture with excipients, or in the form of elixirs, solutions orsuspensions containing flavouring or colouring agents, or they can beinjected parenterally, for example intravenously, intramuscularly orsubcutaneously. For parenteral administration, the compositions may bebest used in the form of a sterile aqueous solution which may containother substances, for example enough salts or monosaccharides to makethe solution isotonic with blood. For buccal or sublingualadministration the compositions may be administered in the form oftablets or lozenges which can be formulated in a conventional manner.

Administration

Typically, a physician will determine the actual dosage which will bemost suitable for an individual subject and it will vary with the age,weight and response of the particular patient and severity of thecondition. The dosages below are exemplary of the average case. Therecan, of course, be individual instances where higher or lower dosageranges are merited.

The compositions (or component parts thereof) of the present inventionmay be administered orally. In addition or in the alternative thecompositions (or component parts thereof) of the present invention maybe administered by direct injection. In addition or in the alternativethe compositions (or component parts thereof) of the present inventionmay be administered topically. In addition or in the alternative thecompositions (or component parts thereof) of the present invention maybe administered by inhalation. In addition or in the alternative thecompositions (or component parts thereof) of the present invention mayalso be administered by one or more of: parenteral, mucosal,intramuscular, intravenous, subcutaneous, intraocular or transdermaladministration means, and are formulated for such administration.

By way of further example, the pharmaceutical composition of the presentinvention may be administered in accordance with a regimen of 1 to 10times per day, such as once or twice per day. The specific dose leveland frequency of dosage for any particular patient may be varied andwill depend upon a variety of factors including the activity of thespecific compound employed, the metabolic stability and length of actionof that compound, the age, body weight, general health, sex, diet, modeand time of administration, rate of excretion, drug combination, theseverity of the particular condition, and the host undergoing therapy.

The term “administered” also includes but is not limited to delivery bya mucosal route, for example, as a nasal spray or aerosol for inhalationor as an ingestable solution; a parenteral route where delivery is by aninjectable form, such as, for example, an intravenous, intramuscular orsubcutaneous route.

Hence, the pharmaceutical composition of the present invention may beadministered by one or more of the following routes: oraladministration, injection (such as direct injection), topical,inhalation, parenteral administration, mucosal administration,intramuscular administration, intravenous administration, subcutaneousadministration, intraocular administration or transdermaladministration.

Diseases

The present invention provides a method for treating and/or preventing adisease in a subject.

The subject may be an animal, preferably a companion animal, mostpreferably a cat or a dog. Preferably the subject is a dog.

The compositions of the invention are contemplated to exhibittherapeutic and/or prophylactic activity, for example, in the treatmentand/or prophylaxis of tumors or other diseases associated with cellproliferation, infections and inflammatory conditions. The compositionsare particularly effective for cancer immunotherapy.

In particular the compositions are effective for immunotherapy of thefeline cancers shown in Table 1 and the canine cancers shown in Table 2.In a highly preferred embodiment there is provided a method for treatingand/or preventing a mammary tumor in a dog.

In general the pharmaceutical compositions of the invention may be usedin the treatment of disorders such as those listed in WOA-98/09985.

The invention is further described, for the purposes of illustrationonly, in the following examples in which reference is made to thefollowing Figures.

FIGURES

FIG. 1 shows human and canine placental sections stained with Y1 ananti-5T4 antibody. A) Human placenta stained with Y1, b) Canine placentanegative control, c) canine placenta stained with Y1

FIG. 2 shows western blotting of truncated human and canine 5T4.

FIG. 3 shows a comparison of feline (SEQ ID NO:3), canine (SEQ ID NO:1),human (SEQ ID NO:12) and murine (SEQ ID NO:11) amino acid sequences.

FIG. 4 shows 293T cells transiently transfected with pIRESneo (A) orpIRES c5T4 (b), formalin fixed and stained with anti-5T4 peptideantiserum.

FIG. 5 shows western blotting of cell supernatants to demonstrate thepresence of a pIRES_c5T4 TM-stable transfectant.

FIG. 6 shows silver staining of c5T4TM-EKMycHis.

-   1. MW marker-   2. Purified c5T4TM-EKMycHis (1.2 μg for silver stain or 675 ng for    western blots-   3. MVA-c5T4LacZ-infected CEF cell lysate (cells infected at MOI of 1    and incubated for 24 h)

FIG. 7 shows western blotting a) anti-c-myc, b) Y1.

FIG. 8 demonstrates the ability of both canine and feline cells tosupport expression of 5T4 from recombinant MVA.

-   1 CF2TH (canine thymus)-   2 D17(poodle osteosarcoma)-   3 AKD (foetal feline lung)-   4 CRFK (feline kidney)-   5 293T (human kidney)

FIG. 9 illustrates cloning c5T4 into the MVA transfer vectors

FIG. 10 shows CEFs uninfected (panels A and C) and infected withMVA-c5T41lacZ (panels B and D) and immunostained with Y1 (Panels A andB) or Y3-P3 (Panels C and D).

FIG. 11 shows western blotting of lysates from CEFs infected withMVA-c5T4lacZ CEF only

-   2: MVA_h5T4 (reduced)-   3: MVA_h5T4 (unreduced)-   4: MVA (wt)-   5: MVA-c5T4lacZ (reduced)-   6: MVA-c5T4lacZ (unreduced).

FIG. 12 shows expression of c5T4 in canine thymic cells.

-   1 CF2TH-MVA-h5T4-   2 CF2TH-MVA-c5T4lacZ-   3 CF2TH-MVA (wt)

FIG. 13 shows examples of 5T4 positive canine tumor samples. A) mammarycarcinoma, B) anal aprocrine carcinoma

EXAMPLES Example 1 Production of Production of new anti-5T4 antibodies

Antibody preparations were raised in chickens against a pool of three,20 amino acid, 5T4 peptides. Regions that are likely to be surfaceexposed (hydrophilic), flexible and charged are good candidates forimmunogenic peptides. The peptides chosen were as follows:

Pep1

CRYEINADPRLTNLSSNSDV (SEQ ID NO:8)

Pep2

CLNHIVPPEDERQNRSFEG (SEQ ID NO:9)

Pep3

NLSGSRLDEVRAGAFEHLPSLRC (SEQ ID NO:10)

One such antiserum, Y1, was demonstrated to recognise both human andcanine 5T4, expressed on placenta (FIG. 1)

Y1 antibody preparation was used for most of the analysis of canine andfeline tissue samples and in the analysis of 5T4 expressing cell linesand the recombinant MVA.

A concern with Y1 was higher than desired background staining. In anattempt to reduce this, affinity purification was carried out usingindividual columns of each of the 3 original peptides used in theimmunisations. It was demonstrated that the majority of antigenicactivity was directed against peptide 3 and so this was used for furtherpurification. The antibody preparation Y3 was successfully purified andsignificantly improved (see below) using this approach.

Western blotting of Myc-His tagged, truncated human and canine 5T4demonstrates that peptide 3 purified Y3 antiserum (Y3-P3) resulted in ahigh activity anti-5T4 antiserum with low background (FIG. 2).(Conversely, purification of Y1 by this method appeared to result in aloss of activity).

Example 2 Isolation of a canine and feline 5T4 genes

A canine genomic library in λ dash was obtained from Stratagene andscreened according to manufacturers instructions using a radiolabelledprobe derived from the human 5T4 cDNA. A number of clones were Isolatedand purified to homogeneity. The gene was identified by southern blot,subcloned into pBSII and sequenced. The canine gene was then used toprobe a feline genomic library in the same way and a clone was isolated,subcloned and sequence. A comparison of the feline, canine, human andmurine amino acid sequences is shown in FIG. 3.

Cloning into Expression Vectors

The full length c5T4 coding region was amplified by PCR from a pBSIIsubclone and cloned into the pIRES neo expression vector (Clontech)

293T cells were transfected with pIRES_c5T4, pIRES_h5T4 and pIRES neo.Transfected cells were immunostained with the H8 mAb and the anti 5T4peptide antiserum, Y1. The Y1 was able to recognise both human andcanine 5T4. Transfected cells were also formalin fixed (see FIG. 4).

Example 3 Cloning of Truncated c5T4 With Optimised Kozac Sequence

To produce the c5T4 protein in sufficient quantities for downstreamapplications such as ELISA, a truncated version of the gene, lacking thetransmembrane region and cytoplasmic tail, but tagged c-terminally witha c-myc epitope and 6 histidines (Myc-His), was constructed by PCR togive a secreted protein that could be readily purified from ChineseHamster Ovary (CHO) cells.

PCR

A 5′ primer incorporating a consensus Kozak sequence was used inconjunction with a 3′ primer situated immediately upstream of thetransmembrane region to amplify the truncated cDNA. This product wasthen spliced into a pGEM-TEasy vector containing an Enterokinasecleavage site N-terminal to a Myc-His tag. The truncated c5T4_EKMycHiswas then cloned into pIRES neo.

This construct (pIRES_c5T4 Tm-)was transfected into CHO cells and stablelines made using G418 selection. Clones expressing the c5T4_EKMycHiswere assessed by immunostaining with the Y1 chicken antiserum and ananti-His antibody.

Western blotting of cell supernatants with an anti-Myc antibodydemonstrates the presence of 5T4 in the cell supernatants of apIRES_c5T4 TM-stable transfectant (FIG. 5).

A suitable clone was then expanded and the supernatant harvested. Thec5T4_EKMycHis protein was then isolated on a nickel column and purityassessed by PAGE followed by coomassie staining and western blotanalysis.

Purification of c5T4TM-EKMycHis

c5T4TM-EKMycHis was double-purified from 1I CHO-c5T4TM-EKMycHissupernatant cells using a 5 ml HiTrap Chelating column and associatedHisTrap kit (Amersham Pharmacia Biotech) and the imidazole concentrationfrom elution was reduced by dialysis against 1× PBS (FIG. 6).

Example 4 Cloning Feline 5T4

The feline 5T4 gene was isolated from a feline genomic lambda library,and following restriction analysis and Southern blotting, a 3 kb DNAfragment was cloned into pBluescript (Stratagene) and sequenced—found tocontain f5T4 gene. This was then amplified by PCR to clone bothfull-length and TM-f5T4 into expression vectors (pIRESneo) fortransfection into CHO cells. The feline gene has also been cloned intothe appropriate MVA transfer plasmids in order to make recombinantMVA_f5T4.

Analysis of cloned cell line CHO-f5T4tm-reveals immunostaining identicalto that of CHO-c5T4tm-.

Example 5 Production of Recombinant MVA_c5T4

The canine 5T4 gene was cloned into the MVA vector and expression of thecanine 5T4 protein in cells infected with the modified virus wasdemonstrated. Expression of the canine protein in canine thymic cellswas also demonstrated and a stock of MVA_c5T4 vaccine prepared.

To demonstrate the ability of both feline and canine cells to supportexpression of 5T4 from recombinant MVA, western blot analysis of thefollowing cells; C2fTH (Canine thymus), D17 (Poodle osteosarcoma), AK-D(Feline Foetal lung), CRFK (Feline Kidney), infected with recombinantMVA_h5T4 was carried out. The cells were infected with MVA or MVA_(h)5T4at an MOI of 5 and then harvested 24 hours later. Lysates were subjectedto PAGE and electroblotting to Hybond ECL and 5T4 was then detected withmAb H8 followed by RαM-HRP and ECL (FIG. 8).

Example 6 Cloning c5T4 into the MVA Transfer Vectors

Methods for propagation of MVA, preparation of CEF cells and homologousrecombination are described in WO 00/29428.

The full length c5T4 cDNA, amplified by PCR, was cloned into an MVAtransfer plasmid with the LacZ marker gene (FIG. 9) which was then usedto make recombinant MVA_c5T4_LacZ by cotransfection with MVA into CEFS.The recombinant virus has been purified to homogeneity through 4 roundsof plaque picking, with no background of wild type virus detected.Immunostaining of infected CEF cells with the Y1 chicken antiserumconfirmed the presence of 5T4 in the virus and PCR amplification of a˜350 bp fragment of 5T4 sequence from viral DNA has confirmed the 5T4gene as canine.

Example 7 Induction of c5T4 expression in CEFs and canine thymic cells

Canine 5T4 was expressed in CEFs infected with recombinant MVA-c5T4.CEFs infected with MVA-c5T4lacZ (MOI=0.02) were immunostained with Y1and Y3-P3 (FIG. 10) showing c5T4 expression.

Lysates from CEFs infected with MVA-c5T4lacZ (MOI=1) were analysed byWestern Blot using Y1 (FIG. 11).

Having shown that the recombinant MVA_c5T4 expresses 5T4 protein in CEFsit was also demonstrated that the protein could be expressed in caninecells. FIG. 12 shows a Western Blot of canine thymic (CF2TH) cellsinfected by MVA_c5T4lacZ and MVA_h5T4, detected using Y1.

Example 8 Immunohistochemistry of Canine and Feline Tissues

Normal canine tissues have been stained for 5T4 using the Y1 antiserum,as shown below:

Tissue No. Positive Brain 0/3 Cerebellum 0/3 Heart 0/3 Lung 1/3 Liver2/3 Kidney 3/3 Pituitary 3/3

The positive samples may be due to 5T4 expression but could be due tosome cross reactivity of Y1. To address the later possibility, thepurified Y3-P3 has been used to repeat these studies of normal tissues.To date only some specific cells of the pituitary have stained positivewith Y3-P3 whilst the cancer tissue samples that have been stained withthis antisera have remained positive.

A summary of the Y1 staining of canine and feline cancer samples aregiven in tables 1 & 2. Out of all the different feline tumor typesstudied 23% (6/26) were positive. This rises to 38% (3/8) for mammarytumors only. The percentage of 5T4 positive tumors is higher in thecanine samples; 45% (30/66) for all types. When mammary tumors, whichmake up 42% of all the tumor types presented to Oncodesign, are analysedseparately, 75% (21/28) are 5T4 positive.

FIG. 13 shows two examples of 5T4 positive canine turmoursamples:mammary carcinoma (A) and anal apocrine carcinoma (B)

TABLE 1 FELINE SAMPLES Sex N^(o). +ve N^(o). −ve % +ve Mammary Tumors:-Adenocarcinoma M F 2 4 33 Lymphoma M F 1 0 Recurrence of canalicular(?)M F 1 100 TOTAL Mammary M F 3 5 38 Intra muscular fibromatosis M F 1 100Malignant Fibroblast M F 1 0 Bladder carcinoma M F 1 100 Sarcoma M F 1 0Fibrosarcoma M 2 0 F 6 0 Fibrohistocytoma M 1 0 F 4 ALL CANCERS (n = 26)M 3 0 F 6 17 26 Both 6 20 23

TABLE 2 CANINE SAMPLES Sex N^(o). +ve N^(o). −ve % +ve Mammary Tumors:-(Adeno)carcinoma M F 15 3 83 Epithelial M F 5 1 83 Mesenchymal M F 1 0Intracanalicular(?) M F 1 100 Sarcoma M F 1 0 Cutaneous Metastasis M F 10 TOTAL M F 21 7 75 Anal apocrine gland (+met.) M 1 0 F 1 100 IntestinalAdenocarcinoma M F 1 100 Pre-cancerous mastopathy M F 2 1 67 EpidermoidCarcinoma M 1 100 F 1 100 Dermo-epidermic lesion M F 1 100 Mast cellcancers M 3 0 F 2 0 Bladder carcinoma M F 1 1 50 Osteosarcoma M 1 100 FHepatocarcinoma M 1 0 F Seminoma M 2 0 F Thyroid Carcinoma M F 1 0Lymphoma M 2 0 F 1 0 Myxosarcoma M 1 0 F Fibrosarcoma M 1 0 FFibrohistocytoma M F 1 0 Rhabdomyosarcoma M F 1 0 Malignant melanoma M 50 F Malignant scwannoma M F 1 0 Hemangiopericytoma M 1 0 F 2 0Sertolinoma M 1 0 F ALL CANCERS (n = 66) M 2 18 10 F 28 18 61 Both 30 3645

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inchemistry, biology or related fields are intended to be within the scopeof the following claims.

1. An isolated nucleotide sequence which encodes a canine 5T4polypeptide set forth in SEQ ID NO:1.
 2. The nucleotide sequence ofclaim 1 having the sequence set forth in SEQ ID NO:2.
 3. A vectorcomprising the nucleotide sequence of claim
 1. 4. The vector of claim 3,wherein the vector is a Modified Vaccinia Ankara (MVA) vector.
 5. A kitwhich comprises: (a) a first composition comprising a nucleotidesequence encoding a canine 5T4 antigen, and (b) a second compositioncomprising the vector of claim 3, for simultaneous, separate orsequential administration to a subject.
 6. The kit of claim 5, whereinthe first composition comprises a nucleotide sequence encoding a canine5T4 antigen, wherein the antigen has the amino acid sequence set forthin SEQ ID NO:1.